The liver and kidney excrete from the body a wide array of positively charged organic molecules of physiological, pharmacological and toxicological significance. The first step in the transepithelial secretion of the [unreadable]organic cations[unreadable] (OCs) by tissues in the kidney and liver involves mediated OC uptake from the blood into cells, across the basolateral membrane. This process, the entry step in OC secretion, is mediated by members of the SLC22A family of transport proteins: OCT2 (in the kidney), and OCT1 (in the liver). OCTs are sites of clinically important drug-drug interactions, and genetic polymorphisms of these transporters have been shown to influence both the efficacy and pharmacokinetics of selected drugs. Development of programs for rational drug design will require an understanding of the structure of these proteins. During the course of the current grant cycle of this continuing research program we developed a homology model of OCT2 structure, based upon crystal structures of several related transporters from the Major Facilitator Superfamily of transport proteins. This model, along with models of other SLC22A transport proteins, has provided novel insights into relationships between transporter structure and function. However, confidence in the accuracy of these models will remain modest, at best, until structural and functional predictions based upon these models receive rigorous testing and validation. This proposal describes four sets of related studies that will continue our ongoing examination of the structure and function of the human ortholog of the organic cation transporter, OCT2. (1) Using site-directed mutagenesis and the substituted cysteine accessibility method (SCAM), we will identify points of transition between transmembrane helices and adjacent loop segments that will establish the topology of this protein. (2) Additional SCAM analyses and cross-linking studies will determine the accessibility of residues in the proposed cleft region of the protein, organization of helices associated with the binding cleft, and will test if conformational shifts of the transporter change the binding surface of the cleft. (3) Proteomic methods (photoaffinity labeling and mass spectrometry) will identify regions in the cleft to which OC substrates bind. (4) The results of these experiments will be integrated with the use of computational methods, including molecular dynamics simulations, to refine the OCT2 model, assess its quality and stability, predict conformational changes associated with the transport process, and characterize ligand interactions with putative binding surfaces. These studies will be essential for development of models that accurately predict and, ideally, preempt unwanted interactions of cationic drugs in both the kidney and liver.